Abstract

Background Over the past 10 years the technique of tryptophan
depletion has been used increasingly as a tool for studying brain serotonergic
systems.

Aims To review the technique of tryptophan depletion and its current
status as a tool for investigating psychiatric disorders.

Method Systematic review of preclinical and clinical studies.

Results Tryptophan depletion produces a marked reduction in plasma
tryptophan and consequently brain serotonin (5-HT) synthesis and release. In
healthy volunteers the effects of tryptophan depletion are influenced by the
characteristics of the subjects and include some mood lowering, some memory
impairment and an increase in aggression. In patients with depression
tryptophan depletion tends to result in no worsening of depression in
untreated subjects but a relapse in those who have responded to
antidepressants (particularly serotonergic agents). In panic disorder the
results are similar.

Conclusions The findings that tryptophan depletion produces a
relapse of symptoms in patients with depression and panic disorder who have
responded to treatment with antidepressants suggests that enhanced 5-HT
function is important in maintaining response in these conditions.

Serotonin (5-HT) is known to play an important role in a wide variety of
functions, including mood, anxiety, aggression, sleep, appetite and sexual
function, although the precise details of the mechanisms involved in these
processes have not been established clearly. The situation has been hampered
by the fact that until very recently it has not been possible to measure brain
5-HT directly, which means that we have had to rely on evidence from indirect
sources. Over the past 10 years, neurotransmitter depletion paradigms have
provided another means of examining the systems involved, and tryptophan (TRP)
depletion in particular has emerged as an important tool for investigating
5-HT function. This review discusses the evolution of the technique of TRP
depletion, the effects that it produces in healthy volunteers and patients
suffering from different disorders, and what this tells us about the role of
5-HT in the pathogenesis and treatment of several psychiatric conditions.

THE TECHNIQUE OF TRYPTOPHAN DEPLETION

The aim of TRP depletion is to lower brain 5-HT by depleting the body of
its amino acid precursor TRP.

Figure 1 illustrates the
processes involved in the transport of TRP into the brain and its synthesis
into 5-HT. A key factor in this is the level of TRP in plasma, which depends
on the balance between the dietary intake of TRP and its removal from the
plasma as a result of protein synthesis. Most of the TRP in plasma is
protein-bound, with only about 5% being left free and available for transport
into the central nervous system (CNS). It is this free TRP that is transported
into the brain across the blood—brain barrier by an active protein
shuttle for which five other large neutral amino acids (LNAAs: valine,
leucine, isoleucine, phenylalanine and tyrosine) also compete. Once in the
brain, TRP can take part in 5-HT synthesis, which is a two-step process.
Tryptophan is first converted into 5-hydroxytryptophan (5-HTP) by the enzyme
tryptophan hydroxylase; 5-HTP is then decarboxylated by the enzyme aromatic
acid decarboxylase to 5-hydroxytryptamine (5-HT). Tryptophan hydroxylase is
only about 50% saturated in the CNS, which means that the rate of 5-HT
synthesis is dependent on the availability of its substrate, free plasma TRP.
This is therefore the rate-limiting step.

Points at which serotonin (5-HT) synthesis may be controlled: (1)
tryptophan availability: dietary restriction; (2) active transport:
competition for the large neutral amino acid protein shuttle; (3) synthesis:
inhibition of tryptophan hydroxylase. Best tryptophan depletion results are
achieved using a combination of control at points 1 and 2. 5-HTP,
5-hydroxytryptophan.

From this description and Fig.
1 it can be seen that three factors are important in determining
the rate of 5-HT synthesis: the total amount of free plasma TRP; how much of
that free TRP crosses the blood—brain barrier; and the activity of the
tryptophan hydroxylase enzyme. Synthesis of 5-HT can be influenced by
interfering with any or all of these factors. Early studies in the 1970s
inhibited tryptophan hydroxylase using the drug parachlorophenylalanine and
reported that this produced a relapse of depressive symptoms in patients who
had previously responded to treatment with imipramine and tranylcypromine.
Parachlorophenylalanine is, however, too toxic for ethical use in human
subjects today, which has meant that interest has focused on techniques
affecting the first two factors. As discussed above, free plasma TRP levels
vary with the amount of dietary TRP and the rate of protein synthesis.
Completely removing TRP from the diet reduces plasma TRP by only 15-20% and
has few behavioural consequences. A much bigger fall in plasma TRP can be
achieved by giving subjects an amino acid load that does not contain TRP, in
the form of a drink. This has two effects: it stimulates protein synthesis in
the liver, which uses up plasma TRP; and the amino acids that are given
include the other LNAAs, which compete with TRP for transport across the
blood—brain barrier and thus restrict the entry of TRP into the brain.
Techniques that influence both of these factors have been shown to produce
maximal brain TRP depletion: that is, using a combination of a low-protein
(low-TRP) diet and a TRP-deficient protein load containing large amounts of
the other LNAAs (reviewed in Reilly et
al, 1997).

The TRP depletion paradigm is based on the hypothesis that reducing plasma
TRP produces a consequent reduction in brain 5-HT synthesis and release. This
has been confirmed in animals, with a large body of preclinical data showing
that depletion of TRP specifically reduces brain 5-HT content and function.
Ingestion of TRP-free amino acid mixtures in laboratory animals leads to
extremely rapid changes in plasma TRP and brain 5-HT content, with maximal
reductions of brain 5-HT occurring within 2 h. It also alters behavioural
indices of 5-HT function (increasing pain sensitivity, acoustic startle, motor
activity and aggression, and reducing rapid eye movement (REM) sleep), effects
that are reversed once brain TRP is restored. In monkeys, TRP depletion has
similar effects, decreasing plasma TRP and cerebrospinal fluid (CSF) TRP and
5-hydroxyindoleacetic acid (5-HIAA).

In humans, the effects of TRP depletion on brain 5-HT have been more
difficult to establish. Neuroendocrine studies have shown that TRP depletion
produces postsynaptic receptor supersensitivity, presumably secondary to
reduced synaptic availability of 5-HT. Recent studies in healthy volunteers
involving continuous CSF sampling have shown that TRP depletion reduces
central TRP and 5-HIAA levels by 80-90% and 24-40%, respectively
(Carpenter et al,
1998; Williams et al,
1999). The time course of these responses was also slightly
delayed compared with the plasma findings, with the nadir for CSF TRP being
7-10 h after TRP depletion and even longer (12-14 h) for 5-HIAA. One study of
the rate of 5-HT synthesis in vivo using positron emission tomography
(PET) has reported that TRP depletion reduces the rate of synthesis by up to
40% of baseline values (Nishizawa et
al, 1997).

Although the details vary, a common procedure for TRP depletion involves
two test days about 1 week apart in a double-blind, crossover design. On both
occasions subjects follow a low-TRP (160 mg/day) diet for 24 h prior to the
test day, followed by an overnight fast extending throughout the test day. On
one test day, subjects consume a drink containing a 100-g load of 15 amino
acids (including the LNAAs described above) that does not contain TRP, and on
the other they have a nutritionally balanced drink that does contain TRP. With
this technique, free (and total) plasma TRP levels are reduced by about 80%
5-7 h after the depleting drink (Delgado
et al, 1990; Williams
et al, 1999). Behavioural measures are recorded
throughout the test day, with peak effects usually being seen 5-7 h after TRP
depletion (i.e. at the point of maximum depletion of plasma TRP).

CLINICAL EFFECTS OF TRYPTOPHAN DEPLETION

Healthy volunteers

The known link between 5-HT and affect led to the early studies of TRP
depletion in healthy volunteers, focusing particularly on mood effects. They
used male subjects exclusively and showed that TRP depletion produced
significant mood lowering (although never amounting to clinical depression) in
these groups (Young et al,
1985; Smith et al,
1987). Subsequent studies, however, have tended to report that TRP
depletion results in no mood effects
(Benkelfat et al,
1994; Carpenter et
al, 1998; Knott et
al, 1999). The discrepancy between these findings is probably
explained by differences in the baseline mood states of subjects in the
studies: those that reported a mood-lowering effect used volunteers with mean
baseline depression scores at the upper end of the normal range, whereas the
more recent negative findings were obtained with fully euthymic subjects.
Studies that have included women have reported a gender difference, with a
tendency for TRP depletion to produce mood lowering in healthy women but not
men, despite similar degrees of plasma TRP depletion. This suggests that women
may be more predisposed to the effects of TRP depletion than men. This is
interesting given the increased incidence of depression in women and evidence
from PET imaging studies suggesting that there may be gender differences in
5-HT metabolism with TRP depletion producing greater biochemical effects in
women than in men (Nishizawa et
al, 1997).

An increased vulnerability to mood alteration during TRP depletion has been
reported in male and female euthymic subjects who have a family history of
affective illness (Benkelfat et
al, 1994; Klaassen et
al, 1999), although this finding has not been replicated by
all studies (Ellenbogen et al,
1999). This suggests that a subgroup of patients might have a
trait that makes them particularly vulnerable to alterations in 5-HT function.
It also fits with clinical experience that a family history of affective
disorder is a predisposing factor for depression.

Studies examining the effects of TRP depletion on anxiety symptoms in
healthy volunteers have reported no significant effects on anxiety
(Klaassen et al,
1998). It does, however, produce an exacerbation in anxiety
(although never amounting to panic) when TRP depletion is combined with
pharmacological panicogenic challenges such as yohimbine
(Goddard et al, 1995)
and CO2 (Klaassen et
al, 1998), which are known to induce panic symptoms in
patients with untreated panic disorder.

Other studies that have focused on the memory and cognitive effects of TRP
depletion in human subjects have shown that TRP depletion specifically impairs
long-term memory formation and interferes with the process of memory
consolidation (Schmitt et al,
2000). Tryptophan depletion has also been reported to result in an
improvement in measures of focused attention
(Schmitt et al,
2000), which may be a result of the inhibitory effect of 5-HT on
other neurotransmitters, particularly those involved with attentional
processes (noradrenaline and acetylcholine). Interestingly, TRP depletion does
not produce changes in other measures of frontal functioning
(Schmitt et al,
2000).

The role of 5-HT in aggressive behaviour has been the subject of much
research. Decreased central 5-HT function has been implicated in impulsive
aggressive acts, including violent offending, arson and suicide. Animal
studies have confirmed these findings and have shown that reducing 5-HT
function increases measures of aggression. Studies in humans have shown that
in healthy volunteers TRP depletion produces a marked rise in the ratings of
aggression during provocation in subjects with high-trait aggression but has
little effect in those with low-trait aggression
(Bjork et al,
2000).

The role of 5-HT in sleep is complex. It increases the number of wakings
during the night and results in changes to sleep architecture, i.e. the
suppression of REM and slow-wave sleep. A large body of work has examined the
sleep findings seen in depression. The classical abnormalities reported
include measures of sleep continuity (difficulty getting off to sleep, waking
during the night and waking early in the morning) and architecture (reduced
REM latency and an increased duration of the first REM period). It was
hypothesised that TRP depletion in healthy volunteers would produce a
reduction in 5-HT function that would be reflected by changes to the sleep
electroencephalogram (EEG), possibly producing changes similar to those seen
in patients with depression. One study reported findings consistent with this,
that is, reduced REM latency after TRP depletion
(Bhatti et al, 1998),
although another study reported no change to REM latency or amount
(Voderholzer et al,
1998).

Most of the studies of TRP depletion have been in the field of depression.
This reflects the intense interest over the past 30 years in the involvement
of 5-HT in the pathogenesis and treatment of affective disorders. At present,
the evidence from untreated patients suggests that 5-HT function is reduced in
depression. The evidence from treated patients suggests that antidepressants
work in depression by increasing 5-HT and/or noradrenaline neurotransmission.
Despite this knowledge, the site of the abnormality/abnormalities causing the
5-HT dysfunction in depression and the precise mechanisms of action of
antidepressants have not been established. The hypotheses include factors such
as the level of TRP availability, the rate of 5-HT synthesis, reuptake or
metabolism, the functioning of pre- or post-synaptic 5-HT receptors and an
altered interaction between 5-HT and other neurotransmitter systems.
Tryptophan depletion provides a way of testing some of these theories.

The effects of TRP depletion in untreated depression were somewhat
unexpected. It was hypothesised that if decreased 5-HT function were
associated with depression, then TRP depletion would lead to a transient
worsening of symptoms. In a seminal study, 43 patients with untreated
depression underwent TRP depletion
(Delgado et al,
1994). This was found to result in no further mood lowering over
the course of the day but, interestingly, a bimodal response the next day,
with 37% describing an improvement in symptoms and 23% a worsening. The lack
of effect of TRP depletion in causing a worsening of depressive symptoms in
untreated patients has been replicated by two other studies (Price et
al, 1997,
1998). One explanation for
this is that 5-HT function is already so diminished that further lowering has
little effect on symptom exacerbation. It may be, however, that deficient 5-HT
function is not the primary or sole cause of depression and may be the result
of dysfunction in regions or circuits in the brain that are modulated by 5-HT.
This would mean that altering 5-HT function by itself would not be expected to
produce immediate effects. The improvement in symptoms on the day after TRP
depletion has been replicated by one study
(Price et al, 1998).
The explanation for this is that TRP depletion results in an up-regulation of
post-synaptic 5-HT receptors because of the decreased release of 5-HT at the
synapse. When 5-HT levels are restored (by the next day), the net effect is an
enhancement of 5-HT function, resulting in the improvement of symptoms.

The role of 5-HT in the mechanism of action of antidepressants in
depression has been examined extensively using TRP depletion. The hypothesis
for these studies was that if, as proposed, a time-dependent process of
neuronal adaptation led to an enhancement of 5-HT neurotransmission after
long-term antidepressant treatment, then acute depletion would be expected to
rapidly reverse the beneficial effects. This has now been tested in multiple
studies that have examined the effects of TRP depletion in patients who had
responded to antidepressant treatments (particularly serotonergic
antidepressants) (Table 1)
(Delgado et al, 1990;
Bremner et al, 1997;
Åberg-Wistedt
et al, 1998; Leyton
et al, 2000). These showed that rapid TRP depletion led
to a return of depressive symptoms in patients who had responded to treatment
with antidepressants. Interestingly, the content of the depressive
symptomatology was often very similar to the original presentation of the
illness and typically occurred about 4-6 h after the depleting drink (i.e. at
the time of peak depletion of plasma TRP).

The rates of relapse from these studies (as measured by an increase in
depressive symptoms on the Hamilton Rating Scale for Depression, HRSD;
Hamilton, 1960) were
consistently higher for those on serotonergic drugs compared with those
receiving more noradrenergic agents. One study that specifically examined this
difference reported that 53% of patients who had responded to fluoxetine
relapsed compared with 7% of patients who had responded to treatment with
desipramine (Delgado et al,
1999). In contrast, catecholamine depletion studies (which use
α
-methyl-p-tyrosine to block the enzyme tyrosine hydoxylase and
thus the synthesis of noradrenaline and dopamine) result in a similar
transient depressive relapse in patients who had been treated successfully
with noradrenergic drugs but not in those on serotonergic antidepressants
(Miller et al, 1996).
This suggests that increasing 5-HT or noradrenaline neurotransmission is
important for the specific function of different antidepressants.

The rates of relapse for patients on selective serotonin reuptake
inhibitors (SSRIs) ranged in the different studies from 80%
(Delgado et al, 1991)
in patients remitted for at least 2 weeks, to only 30%
(Bremner et al, 1997)
in patients remitted for an average of 45 weeks. This suggests that the TRP
depletion-induced relapse may be more likely in recently remitted patients
compared with those who have been treated for longer periods of time. It may
be that increasing synaptic 5-HT is only the first step in a cascade of events
that lead to adaptive changes in 5-HT neurons, and that disrupting 5-HT
function at this later stage has less effect.

The time course of the effects of depletion paradigms (i.e. over hours)
implies that synaptic availability of 5-HT is more important than
post-synaptic receptor effects, which would not be expected to change as
rapidly. This is quite a major shift in thinking, because previous teaching
has suggested that the delay in the clinical effect of antidepressants is
explained by changes to postsynaptic receptors and other processes downstream
of the synapse.

Two studies have used PET imaging to examine the effects of TRP depletion
in remitted patients. Both reported that TRP depletion resulted in a decrease
in brain metabolism in the areas of the brain implicated in the pathogenesis
of depression (i.e. dorsolateral prefrontal and orbitofrontal cortex, amygdala
and thalamus) (Bremner et al,
1997; Morris et al,
1999). Interestingly, in one study changes were seen only in 7/21
patients and were limited to those who relapsed as a result of TRP depletion
(Bremner et al,
1997).

Patients who have had previous episodes of depression are known to be
vulnerable to further episodes and it might be expected that TRP depletion in
this group would result in relapse of depressive symptoms. Three studies have
examined this specifically, using subjects with a past history of depression
who at the time were well and on no medication
(Leyton et al, 1997;
Smith et al, 1997;
Moreno et al, 1999).
Two of the three studies reported greater rates of depressive relapse after
TRP depletion than after the control drink
(Smith et al, 1997;
Moreno et al, 1999).
These findings imply that deficient 5-HT function may play a part in
triggering depressive episodes in patients with a vulnerability or
susceptibility to changes in 5-HT function.

The sleep effects of TRP depletion in patients with remitted depression are
interesting. One study has reported that although none of a group of ten men
remitted for 2-13 months relapsed during TRP depletion, their sleep EEGs did
regain some of the abnormalities characteristic of depression (i.e. reduced
sleep and REM latencies and increased REM percentage and density)
(Moore et al, 1998).
This suggests that sleep may be a sensitive index of TRP levels.

Seasonal affective disorder (SAD) is a subtype of mood disorder
characterised by recurrent major depressive episodes that occur regularly in
the autumn and winter and respond to light therapy. The pathophysiology of SAD
and the mechanism of action of light therapy are still poorly understood, but
several lines of evidence suggest that 5-HT may be important
(Joseph-Vanderpool et al,
1993). The results of TRP depletion in this group are similar to
those reported with depression. Untreated patients are the least vulnerable to
the mood-lowering effects of TRP depletion
(Neumeister et al,
1997b). Patients with SAD who have been treated and are
in remission after light therapy are likely to relapse after TRP depletion
(Lam et al, 1996;
Neumeister et al,
1997a). Long-term remitted patients produce mixed
responses to TRP depletion, with one study reporting relapse rates of 73%
(Neumeister et al,
1998) and another reporting no effect on symptoms
(Lam et al,
2000).

Anxiety disorders

Serotonin neurotransmission is thought to play a central role in panic and
anxiety in general. Perhaps the strongest evidence comes from treatment
studies, which have shown that many of the disorders, including panic, social
phobia and obsessive—compulsive disorder (OCD), are amenable to
treatment with serotonergic drugs such as the SSRIs (discussed by
Bell & Nutt, 1998).

Most TRP depletion studies in anxiety have been performed in panic disorder
and OCD, although there are ongoing studies in social phobia and generalised
anxiety disorder.

Panic disorder

Patients with untreated panic disorder experience panic symptoms in
response to panicogenic challenges (e.g. lactate, CO2,
cholecystokinin-4, yohimbine or flumazenil), a reaction that is blocked by
effective antidepressant treatment of the condition. Studies consistently show
that in untreated patients TRP depletion alone produces no exacerbation of
anxiety (Miller et al,
2000). However, when TRP depletion is combined with a panicogenic
challenge it tends to result in an increase in anxiety and an increased rate
of panic attacks (Miller et al,
2000), although one study reported a reduction in the expected
number of panic attacks compared with normal controls
(Schruers et al,
2000). We have reported recently on the effects of TRP depletion
and a flumazenil challenge in patients who have responded to treatment with
SSRIs (Nutt et al,
1999). We showed that 5/8 patients panicked in response to
flumazenil when they were TRP depleted, compared with 0/8 on the control day.
This suggests that enhanced synaptic 5-HT neurotransmission is important in
maintaining the response to SSRIs in this condition; and when this is reduced
(by TRP depletion), panic results (a finding similar to that reported in
depression).

Obsessive—compulsive disorder

Serotonergic tricyclic antidepressants and SSRIs have been used
successfully in the treatment of OCD. It was hypothesised that, as with
depression and panic disorder, TRP depletion might be expected to exacerbate
OCD in this group of patients. Four studies have examined the effects of TRP
depletion in OCD in remitted patients either taking medication
(Barr et al, 1994) or
remitted and drug-free (Smeraldi et
al, 1997; Huwig-Poppe
et al, 1999). These have all shown that TRP depletion
produces no effect on OCD or Tourette symptoms, although some mood-lowering
changes were reported. The difference in this response compared with the
findings in depression and panic disorder suggests that the treatment of OCD
may be less dependent on the synaptic availability of 5-HT than these other
conditions, and more dependent on changes further downstream. Alternatively,
it may be a reflection of the design of these studies, which did not combine
TRP depletion with a challenge (as used in the panic disorder studies); and it
may be that if this were done, TRP depletion would produce a relapse in
symptoms.

Eating disorders

There is considerable evidence for a role for 5-HT in the control of
feeding behaviour, with a reduction in 5-HT function being shown to lead to
impaired satiety and weight gain in animals. Dieting is known to lower 5-HT
function in healthy women (Anderson et
al, 1990) and to increase their prolactin responses to TRP
(implying post-synaptic supersensitivity as a result of reduced synaptic
availability). It has also been reported that women with a personal history of
depression become more depressed during dieting than those without
(Smith et al,
1999).

Bulimia nervosa has been linked with lowered 5-HT function. Studies in
acutely ill untreated subjects with bulimia nervosa found increases in
subjective ratings of the fear of fatness and binge eating as a result of TRP
depletion (Kaye et al,
2000). A similar study in a group of ten patients in remission
from the condition reported that TRP depletion produced an increase in the
symptoms of fear of fatness, fear of losing control over eating and an
increase in the urge to eat (Smith et
al, 1999). In both untreated and treated patients with
bulimia nervosa and those in remission, there was a vulnerability to the
mood-lowering effects of TRP depletion.

To date, no TRP depletion studies have been performed in anorexia
nervosa.

Other disorders

Dementia

Studies that examined the effects of TRP depletion on memory and related
cognitive processes have provided some evidence that reducing 5-HT function
through this technique impairs memory consolidation. This may be of particular
relevance for disorders in which a deficit of 5-HT function is known to be
linked with cognitive effects (e.g. Alzheimer's disease, where diminished 5-HT
levels were found, at post-mortem). In a study that examined the effects of
TRP depletion in patients with Alzheimer's disease, a significant impairment
in cognitive functioning was reported, again suggesting that compromised
serotonergic function (in combination with cholinergic deficit) may make an
important contribution to the cognitive decline seen in this group of patients
(Porter et al,
2000).

Bipolar disorder

A role for serotonin in mania has been hypothesised
(Yatham et al, 1999),
although to date no TRP depletion studies have been performed in patients with
acute mania. In patients recovered from bipolar disorder, TRP depletion has
been reported to have either no effect
(Cassidy et al, 1998)
or to cause a slight but insignificant (due to small sample size) relapse of
mania (Cappiello et al,
1997).

Schizophrenia

There is some evidence that 5-HT may be at least partially responsible for
the negative symptoms of schizophrenia. The effects of TRP depletion in
schizophrenia, however, appear to be quite complex. In one study, TRP
depletion exacerbated depressive symptoms and had no effects on positive or
negative psychotic symptoms (D'Souza
et al, 1994), whereas in another study it worsened
negative symptoms (Sharma et al,
1997). In a recent study of neuroleptictreated patients with
schizophrenia, TRP depletion resulted in an improvement on two measures of
cognition (Speed and Comprehension of Language Processing and Wisconsin Card
Sorting Test; Golightly et al,
1999).

DISCUSSION

Tryptophan depletion provides a way of examining the role of 5-HT in the
pathophysiology of several psychiatric disorders and the mechanism of action
of medication (antidepressants in particular). The responses produced by TRP
depletion in different patient groups give us some information about the role
of 5-HT in these disorders. Many questions remain unanswered but TRP depletion
does provide an interesting and usable tool with which to examine the
involvement of 5-HT in these conditions.

Clinical Implications and Limitations

CLINICAL IMPLICATIONS

Findings that tryptophan (TRP) depletion produces a relapse of symptoms
in treated depression (and panic disorder) suggest that enhanced serotonin
(5-HT) function is important in maintaining response to
antidepressants.

Findings that TRP depletion produces relapse or precipitates symptoms in
patients with a personal or family history of affective disorders suggest that
these subjects have a vulnerability to alterations in 5-HT function.

Lack of effect of TRP depletion in untreated depression and panic
disorder and in healthy volunteers implies that 5-HT dysfunction is not the
primary or sole cause of these disorders.

LIMITATIONS

We are not yet able to use TRP depletion as a tool to predict the
likelihood of relapse when patients are taken off antidepressants.

In humans it is difficult to establish the degree to which TRP depletion
produces a reduction in brain 5-HT and the time course of this
response.

There are only a small number of studies in conditions other than
depression, which limits the conclusions that can be made about these
conditions.

Acknowledgments

We thank Dr Spilios Argyropoulos for his help with revisions to this
paper.

Nishizawa, S., Benkelfat, C., Young, S. N., et al
(1997) Differences between males and females in rates of
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